Aminoisobutyric acid (3-amino-2-methylpropionic acid) is a chiral molecule and exists in the form of the (R) and the (S) enantiomers. (R) (xe2x88x92) Aminoisobutyric acid is a useful intermediate in the convergent synthesis of cryptophycin molecules; see for example, Barrow, R. A., et. al, J. Am. Chem. Soc. 117, 2479-2490 (1995). Accordingly, aminoisobutyric acid, for use in the synthesis of cryptophycin, must be synthesized in a chiral form, or synthesized as a racemic molecule and resolved.
Cryptophycins are potent tumor-selective cytotoxins which are elaborated by terrestrial blue green algae. They show excellent activity against solid tumors implanted in including drug resistant tumors, and are thus useful as anti-cancer agents. Cryptophycins were originally isolated from blue green algae. However, they have now been synthesized using a convergent synthesis in which the cryptophycin molecule is put together from four sub-units, named in the literature A, B, C and D. For one group of cryptophycins, the C Unit is R(xe2x88x92)aminoisobutyric acid. Barrow (J. Am. Chem. Soc., 117, 2479-2490 (1995)) reports a synthesis of (R) (xe2x88x92)3-amino-2-methylpropionic acid beginning with the chiral molecule, methyl(S)-(+)-3-hydroxy-2-methylpropanoate which is a rather expensive starting material. The synthesis begins with the ammonolysis of the propanoate ester at 50xc2x0 C. in a sealed tube for one week. The ammonolysis could be speeded up through the use of 10% sodium cyanide as a catalyst. However, the cyanide catalyst is difficult to remove from the reaction product. In the next step, the amide is reduced with borane to produce an amino alcohol. After protection of the amine, the alcohol is oxidized with ruthenium tetraoxide to give the desired carboxylic acid. This is a complex and expensive synthesis.
A more recent synthesis (Rabida, Res, et al., J. Org. Chem. 61, p.6289(1996)) starts with S-(+)-3-bromo-2-methyl-1-propanol, and produces the desired acid in 70% yield. However, S-(+)-3-bromo-2-methyl-1-propanol is an expensive starting material.
The (S) enantiomer of aminoisobutyric acid occasionally shows up in human serum and urine; see for example, Row, Charles R., et al. MOL. GENET. METAB. (1998), 65 (1, 35-43); Tamaki, Nanaya, Biochim. Biophys. Acta (1990), 1035 (1) 117-19. The pure (S) enantiomer of aminoisobutyric acid may be used as an analytical standard in metabolic studies. Several elegant and expensive synthesis have been reported for the (S) enantiomer of aminoisobutyric acid. Eusedio, Juaristi, et al. (Tetrahedron: Asymmetry (1996), 7(8), 2233-2246) report a synthesis from 1-benzoyl-2 (S)-tert-butyl-3-methylperhydropyrimidin-4-one. M. Akssira (Amino Acids (1994), 7 (1), 79-81) reports the synthesis using the xcex2-alanine derivative with two chiral handles.
The present invention provides the process for resolving racemic 3-amino-2-methylpropionic acid. The amino group of the acid is protected, and the protected acid is reacted with a chiral amine to form diastereomeric salts. The diastereomeric salts are recrystallized from ethyl acetate to separate the salts into pure fractions. The protected acid is recovered from the purified diastereomeric salts by treatment with base, and the amine-protecting group is removed with treatment with a strong mineral acid solution.
The present invention provides a simple and economical method for resolving racemic aminoisobutyric acid into the (S) and (R) enantiomers. A convenient synthesis of racemic aminoisobutyric acid is also disclosed.
For the purposes of the present invention, the racemic aminoisobutyric acid may be synthesized by any convenient method. Such methods are known to those skilled in the art and the racemic acid produced by any method may be readily resolved by the method of this invention. A convenient synthesis of racemic aminoisobutyric acid begins with ethyl 2-cyanopropionate. The cyano portion of the molecule is reduced to an amine with hydrogen gas and a catalyst, to form the ethyl ester of the amino acid. The amino group is protected, and the ester is cleaved to form the amino protected amino carboxylic acid.
Ethyl-2-cyanopropionate may be obtained by careful hydrogenation of ethyl-2-cyanoacrylate, which is readily available and often used as a glue. Ethyl-2-cyanopropionate is reacted with hydrogen in the presence of a hydrogenation catalyst in a suitable solvent such as 1,4 dioxanq, THF, t-butyl-methyl ether. The preferred solvent is THF. The mixture is heated to reflux and subjected to hydrogenation at a moderate pressure in the range of 40-70 pound psia (absolute pressure). Suitable hydrogenation catalysts include platinum, platinum on charcoal, platinum oxide, ranoy nickel and various rhodium catalysts. The preferred catalyst is 5% rhodium on alumina.
It is preferred to conduct the hydrogenation in the presence of a amine protective reagent to protect the amine as it is being formed. Among the protective groups that can be used include BOC (t-butyloxycarbonyl), carbobenzyloxy, phenoxyacetyl, 2,2,2-trichloro-1,1-dimethylethylcarbonyl, and 2,2,2-trichloroethoxycarbonyl. Other suitable protective groups are well known to those skilled in the art. See, for example, Protective Groups in Organic Synthesis, Greene, Theodora W., 1931, 3 rd ed./Theodora W. Greene and Peter G. M. Wuts, New York: Wiley, c1999. After the hydrogenation is complete (in approximately 18-24 hours), the solution is concentrated by the removal of solvent. Water, and a strong inorganic base are added to cause the hydrolysis of the ester portion of the molecule. Suitable inorganic bases include lithium hydroxide, sodium hydroxide, sodium carbonate, potassium hydroxide and potassium carbonate. Finally, the entire mixture is neutralized with a suitable acid including inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid or a simple inorganic acid such as acetic acid, and the amino protected-aminoisobutyric acid is extracted with a solvent and isolated as a low-melting waxy solid. One advantage of conducting the hydrogenation process in the presence of a protective group is that it yields the aminoisobutyric acid with the amino group already protected. If one synthesizes aminoisobutyric acid by some other route in which the acid is formed without a protective group on the amine, it is important that the amine be protected before the resolution step.
The resolution is accomplished by first placing a blocking group or protective group on the amine group of amino isobutyric acid and then by reacting the carboxylic acid group of the amino blocked aminoisobutyric acid with a chiral amine to form diastereomeric salts. Obviously, the amine portion of the aminobutyric acid must be protected so that it will not compete with the reaction between the chiral amine and the carboxylic acid. Suitable amines for reaction with the acid portion of the aminoisobutyric acid include (xcex1-methyl-benzylamine, quinine (the (R) enantiomer of xcex2-methoxycinchonan-9-ol) and quinidine (the (S) enantiomer of xcex2-methoxycinchonan-9-ol). The preferred amine is (xcex1-methyl-benzylamine. Either enantiomer of the amine may be used. The reaction and resolution process using either enantiomer of a chiral amine is illustrated in Scheme II. In this scheme, the enantiomer for the acid is shown in bold face standard type, and the enantiomer for the amine is shown in italics. The (RR) salts and the (SS) salt have higher melting points than the (RS) and the (SR) salts. For xcex1-methyl-benzylamine, the (RR) and (SS) salts melt at 133xc2x0 C. while the (RS) and (SR) salts melt at 119xc2x0 C.
Several solvents were tested for the selective crystallization of the diastereomeric salts. These solvents included ethanol, methanol, THF, t-butylmethyl ether, isopropyl alcohol, and combinations of these solvents. None of these solvents provided the desired separation of the diastereomeric salts.
Surprisingly, it was found that in ethyl acetate, the (RR) and (SS) salts were substantially less soluble than the (RS) and the (SR) salts. When the mixture diastereomeric salts is dissolved in ethyl acetate, the (RR) salt or (SS) salt (depending upon the enantiomer of the amine which has been selected) precipitates at approximately 40xc2x0 C. This precipitate is collected, and the filtrate is cooled at approximately 25xc2x0 C. the (SR) or (RS) salts (depending upon the enantiomer of the amine selected) precipitates.
The precipitated salts are enriched in a preferred enantiomer of the acid. Thus, if the (S) enantiomer of the amine is selected, the high temperature precipitate will be enriched in the (S) enantiomer of the acid. The low temperature precipitate will be enriched in the (R) enantiomer of the acid. If the (R) enantiomer of the amine is selected, then the high temperature precipitate will be enriched in the (R) enantiomer of the acid, and the low temperature precipitate will be enriched in the (S) enantiomer of the acid. The first recrystallization from ethyl acetate produces crystal fractions having ratios of 2:1 in favor of the preferred diasteriomeric salt. Further recrystallizations may be done to produce crystal fractions having the desired level of purity. An enantiomeric purity of 95% is achieved with four recrystallizations.
The racemic 3-amino-2-methylpropionic acid, with an appropriate block for the amine group may be reacted with either the (R) or (S) enantiomer of the selected amine. If one selects the (R) enantiomer, the isolation of the (R) acid will be somewhat easier since the (RR) diastereomeric salt precipitates from the solution before the (RS) diastereomeric salt. One can isolate both the (RR) and the (RS) diastereomeres and selectively recrystallize each one thereby obtaining the two diastereomeric salts at the desired level of purity.
By treating each diastereomeric salt with a solution of an inorganic base such as alkali metal carbonate or hydroxide, or alkaline earth carbonates and hydroxides, one can cause disassociation of the salts, and thereby recover both the (R) and the (S) enantiomers of the acid. However, it is often easier to recrystallize higher melting crystals, that is, the (RR) diastereomeric salt or the (SS) diastereomeric salt. Accordingly, if one desires only one enantiomer of 3-amino-2-methylpropionic acid, it is preferred to start with an amine of the same stereoisomeric family as the desired enantiomer of the acid. Thus, if one desires the (R) enantiomer of the acid, it is preferred to form the diastereomeric salts from an (R) amine. Similarly, if the (S) enantiomer of the acid is desired, it is preferred to form the diastereomeric salts from an S amine.
The acid may be recovered from the amine protected acid by dissolving the amine protected in a suitable solvent such as methylene chloride, and washing the solution with an aqueous mineral acid solution such as 1N hydrochloric acid, 1N sulfuric acid, and 1N phosphoric acid.